Replacement method of influenza A Virus antigenic determinant

A type A influenza virus, antigen-determined technology, applied in the field of bioengineering, can solve the problem of unsatisfactory repeated use of viral vectors and other problems

Inactive Publication Date: 2010-10-13
ZHEJIANG ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Influenza virus vectors escape the tracking of the immune system when they are reused through the replaceme...

Method used

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  • Replacement method of influenza A Virus antigenic determinant
  • Replacement method of influenza A Virus antigenic determinant
  • Replacement method of influenza A Virus antigenic determinant

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Cloning of the hemagglutinin gene

[0052] In a biological safety cabinet, viral RNA was extracted with Trizol according to the method provided by Initrogen Company, and viral cDNA was synthesized by reverse transcriptase MMLV with V13 as a reverse transcription primer. And using the above-mentioned viral cDNA as a template, a full-length HA gene fragment containing complete untranslated regions at both ends was obtained by PCR amplification with two primers V12 and V13. Then the full-length HA gene fragment was used as a template to amplify the full-length HA gene by two primers 5BmHA and 3BmHA, and at the same time, a BsmBI restriction enzyme site was introduced at both ends of the gene. Specifically: the primers used to amplify the full-length HA gene of H1 subtype influenza virus (A / WSN / 1933) are V12 and V13, and then use the amplified product as a template to introduce primers 5BmHA and 3BmHA at both ends of the HA gene A BsmBI restriction enzyme site for cloning ...

Embodiment 2

[0059] The full-length HA gene was cloned into the transcription vector pPolI

[0060] The full-length HA gene with BsmBI restriction enzyme sites at both ends described in Example 1 was digested with BsmBI, and the digested product was purified and connected to the transcription vector pPolI digested by BsmBI, and the recombinant plasmid was screened and carried out. DNA sequence determination. Insert the recombinant plasmid of H1 subtype A / WSN / 1933 (H1N1) strain HA gene in pPolI named pPolI / WSN-HA / H1; insert H3 subtype A / Memphis / 1 / 1971 (H3N2) virus in pPolI The recombinant plasmid of strain HA gene was named pPolI / Memphis-HA / H3; the recombinant plasmid of inserting H5 subtype A / duck / HK / 312 / 78 (H5N3) strain HA gene into pPolI was named pPolI / HK312-HA / H5.

Embodiment 3

[0062] Site-directed mutagenesis, introducing new restriction sites

[0063] By site-directed mutagenesis, a new restriction site was introduced into the recombinant plasmid described in Example 2 for antigen replacement. Taking the introduction of the XbaI restriction enzyme site in the recombinant plasmid pPolI / WSN / H1 as an example, the specific steps are as follows:

[0064] Using pPolI / WSN / H1 as template and H1XbaHA82F and H1XbaHA82R as point mutation primers, PCR amplification was carried out. Amplification conditions were pre-denaturation at 94°C for 3 min; denaturation at 94°C for 45 s, annealing at 55°C for 45 s, extension at 72°C for 6 min, a total of 18 cycles, and a final extension at 72°C for 10 min. After purification, the amplified product was digested with restriction endonuclease DpnI at 37°C for 1 h, precipitated with ethanol / sodium acetate and dissolved in 20 μl double distilled water, and 1 μl was transformed into Escherichia coli DH5α, and the mutant clone...

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Abstract

The invention discloses a replacement method of influenza A virus antigenic determinant. The method aims at the position of antigenic determinant to utilize and design restriction enzyme cutting site, thus realizing that the entire or part of the sequence of antigenic determinant of the strains of influenza in the same subtype or different subtypes can be changed; and an eight-plasmid reverse gene operation system is used to rescue the recombinant influenza virus of which antigenicity is through artificial directed change, and the method has great practical value in the quick preparation of attenuated live vaccine of influenza virus and repeated use of virus vector in gene therapy.

Description

technical field [0001] The invention belongs to the field of bioengineering, and in particular relates to a method for replacing the antigenic determinant region of influenza A virus by replacing the antigenic fragments on the HA gene between different strains of influenza A virus. Background technique [0002] Influenza, called flu for short, has been known for more than 100 years. It becomes popular every autumn, winter and winter and spring, becoming one of the most serious infectious diseases in the world. Several large-scale global epidemics occurred in the 20th century, resulting in the death of a large number of people and bringing serious disasters to human society. For example, the 1918 Spanish flu (H1N1) killed an estimated more than 50 million people, the 1957 Asian flu (H2N2) and the 1968 Hong Kong flu (H3N2) also killed millions more, and the number of deaths from the disease Far exceeds the number of people who died in the war. In 2009, the new type A H1N1 in...

Claims

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Application Information

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IPC IPC(8): C12N15/44C12N15/63C12N15/09C12N7/01
Inventor 郭潮潭沃恩康
Owner ZHEJIANG ACAD OF MEDICAL SCI
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